Varying Flavobacterium psychrophilum shedding dynamics in three bacterial coldwater disease-susceptible salmonid (Family Salmonidae) species

ABSTRACT Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and is responsible for substantial losses in farm and hatchery-reared salmonids (Family Salmonidae). Although F. psychrophilum infects multiple economically important salmonids and is transmitted horizontally, the extent of knowledge regarding F. psychrophilum shedding rates and duration is limited to rainbow trout (Oncorhynchus mykiss). Concurrently, hundreds of F. psychrophilum sequence types (STs) have been described using multilocus sequence typing (MLST), and evidence suggests that some variants have distinct phenotypes, including differences in host associations. Whether shedding dynamics differ among F. psychrophilum variants and/or salmonids remains unknown. Thus, three F. psychrophilum isolates (e.g., US19, US62, and US87) in three MLST STs (e.g., ST13, ST277, and ST275) with apparent host associations for coho salmon (O. kisutch), Atlantic salmon (Salmo salar), or rainbow trout were intramuscularly injected into each respective fish species. Shedding rates of live and dead fish were determined by quantifying F. psychrophilum loads in water via quantitative PCR. Both live and dead Atlantic and coho salmon shed F. psychrophilum, as did live and dead rainbow trout. Regardless of salmonid species, dead fish shed F. psychrophilum at higher rates (e.g., up to ~108–1010 cells/fish/hour) compared to live fish (up to ~107–109 cells/fish/hour) and for a longer duration (5–35 days vs 98 days); however, shedding dynamics varied by F. psychrophilum variant and/or host species, a matter that may complicate BCWD management. Findings herein expand knowledge on F. psychrophilum shedding dynamics across multiple salmonid species and can be used to inform future BCWD management strategies. IMPORTANCE Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, both of which cause substantial losses in farmed and hatchery-reared salmon and trout populations worldwide. This study provides insight into F. psychrophilum shedding dynamics in rainbow trout (Oncorhynchus mykiss) and, for the first time, coho salmon (O. kisutch) and Atlantic salmon (Salmo salar). Findings revealed that live and dead fish of all fish species shed the bacterium. However, dead fish shed F. psychrophilum at higher rates than living fish, emphasizing the importance of removing dead fish in farms and hatcheries. Furthermore, shedding dynamics may differ according to F. psychrophilum genetic variant and/or fish species, a matter that may complicate BCWD management. Overall, study results provide deeper insight into F. psychrophilum shedding dynamics and will guide future BCWD management strategies.


Reaction mixture and cycling parameters
The F. psychrophilum-specific qPCR developed by Marancik and Wiens (30) was per formed according to protocol with some modification.Briefly, each 15-µL reaction mixture contained 7.5 µL of TaqMan Environmental Master Mix 2.0, 0.67 µM of forward and reverse primers, 0.17 µM of TaqMan probe, 0.60 µL of VetMAX Xeno Internal Positive Control (IPC) Assay, and 1 µL of template, with nuclease-free water comprising the remainder.Reactions were run in MicroAmp Optical 96-Well Fast Reaction Plates (0.1 mL) covered with MicroAmp Optical Adhesive Film.A QuantStudio 3 real-time thermal cycler (Thermo Fisher Scientific) was used to amplify the 77-bp target amplicon according to the cycling program of Marancik and Wiens (30).All consumables were purchased through Thermo Fisher Scientific except the primers, which were obtained from Integrated DNA Technologies (IDT).

Preparation of standards
The qPCR target gene sequence was PCR amplified using previously extracted gDNA from F. psychrophilum isolate US53 (20) and the same primers used for qPCR.Briefly, a 50-µL reaction mixture was prepared with 25-µL 2X GoTaq Green Master Mix (Promega), 0.25 µM forward and reverse primers, 40 ng of US53 gDNA, with nuclease-free water comprising the remainder.A touchdown protocol consisting of initial denaturation for 2 min at 94°C followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and a final extension step at 72°C for 7 min was performed using an Eppendorf Mastercycler pro conventional thermal cycler (Thermo Fisher Scientific).The PCR product was run through a 1.5% agarose gel prepared with 1× SYBR Safe DNA gel stain (Thermo Fisher Scientific) for 40 min at 100 V, after which the gel was viewed under UV transillumina tion to confirm the presence of an appropriately sized band.The PCR product (57.1% guanine-cytosine content; molecular weight of 37,497.9g mol −1 ) (30) was purified using the QIAquick PCR Purification Kit (Qiagen) and then quantified using a Qubit fluorometer and the broad range Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific).Gene copy concentration was calculated using a previously published formula (31): Gene copies/μL = ngDNA μL −1 × 6.022 × 10 23 copies mol −1 37, 497.9 g mol −1 × 1 × 10 9 ng g −1 Serial 10-fold dilutions of purified amplicon were made over nine orders of magnitude (e.g., 10 8 copies/μL to 10° copies/μL) using 1X IDTE solution (pH 8.0; Integrated DNA Technologies) supplemented with 100 ng/µL tRNA (Yeast tRNA; Thermo Fisher Scientific).Two standard curve assays were performed, each on individual 96-well plates using eight replicate reactions of each dilution (i.e., qPCR standard); intra-and inter-assay variation was determined using mean quantification cycle (Cq), Cq standard deviation, and coefficient of variation (CV).Assay efficiency, slope, and the correlation coefficient (R 2 ) of each assay were also calculated; efficiency estimates between 90% and 110% were considered acceptable (32,33).These newly generated standards were used to determine F. psychrophilum DNA extraction efficiency, the limit of F. psychrophilum quantification and detection from spiked water (DNA extraction efficiency and limit of quantification and detection section), and to quantify F. psychrophilum loads in 50-mL water samples obtained during the in vivo shedding experiments (Determination of Flavobacterium psychrophilum shedding rate from live and dead fish via qPCR section).

Preparation of mock water samples containing Flavobacterium psychrophilum
Flavobacterium psychrophilum isolate US53 was revived from cryostock (maintained at −80°C) on F. psychrophilum medium A (FPM-A) (34), incubated for 48 hours at 15°C, visually inspected for purity, inoculated into 250 mL of analogous broth, and incubated with constant shaking (180 rpm) for 48 hours at 15°C.Bacteria were harvested via centrifugation (2,571 × g, 15 minutes) and resuspended in 50 mL of ultraviolet light-trea ted, sand-filtered well water (i.e., the same water supplying the shedding experimental aquaria in In vivo assessment of shedding dynamics in Atlantic salmon, coho salmon, and rainbow trout section), which was then serially diluted up to 100,000,000-fold in 10-fold increments to create nine total mock water samples.To quantify bacteria in the most concentrated mock water sample, a 1-mL aliquot was serially diluted 100,000,000-fold in 10-fold increments and plated on FPM-A in duplicate and then incubated for 7 days, after which final colony counts were performed.All mock water samples were brought to a final volume of 50 mL to replicate the water sampling volume used during the shedding experiment.

Bacterial DNA extraction from water
Each mock water sample (n = 9; Preparation of mock water samples containing Flavobacterium psychrophilum section) was vacuum filtered through a single sterile piece of Whatman qualitative filter paper (grade 4, 20-25 μm pore size, 70 mm in diameter; Millipore Sigma) that had been placed in a 70-mm diameter Büchner funnel.The filter paper was removed from the Büchner funnel and placed into a sterile Petri dish; sterile forceps were used to first fold the paper in half (the side receiving the bacterial suspension was facing inward) and then loosely roll it into a cylindrical shape.The rolled paper was placed inside a PowerBead Pro tube of the DNeasy PowerSoil Pro Kit (Qiagen) along with 20,000 copies of Xeno IPC to monitor inhibition; DNA was then extracted according to the manufacturers' protocol, resulting in 50 µL of eluted DNA per dilution.
Flavobacterium psychrophilum DNA extraction efficiency was calculated as the quotient of the mean qPCR-determined concentration of F. psychrophilum (in cells per milliliter) and the mean theoretical input (i.e., pre-DNA extraction) concentration of F. psychrophilum (41).The input concentration of F. psychrophilum was considered theoretical, given that the most concentrated mock water sample alone was quanti fied via plate counts (Preparation of mock water samples containing Flavobacterium psychrophilum section).The median of the mean F. psychrophilum DNA extraction efficiency values was used as the universal DNA extraction efficiency (35,41); this number was used to apply a DNA extraction correction factor (DECF) to all qPCR-derived F. psychrophilum concentrations according to the following formula:

DECF = 100 Median of mean DNA extraction efficiency
The LOD was determined by running 10 replicate reactions theoretically containing 100, 10, and 1 F. psychrophilum cell(s)/mL and defined as the lowest mean qPCR-derived concentration of F. psychrophilum that could be detected in ≥95% of qPCR replicate reactions; this concentration was multiplied by the DECF to yield the LOD (41).
In vivo assessment of shedding dynamics in Atlantic salmon, coho salmon, and rainbow trout

Origin of fish for shedding experiment
Embryonated Atlantic salmon and rainbow trout eggs were sourced from a commercial egg distributor, while embryonated coho salmon eggs were procured from Platte River State Fish Hatchery.Coordination occurred so that all eggs from the three species arrived at the Michigan State University-University Research Containment Facility on the same day.In brief and upon receipt, eggs were disinfected with 100-ppm iodophor solution (pH 7.30) for 10 minutes before being placed in a vertical incubator supplied with UV-treated, sand-filtered well water maintained at 12°C ± 1°C until hatching.Sac-fry were then moved to aerated flow-through tanks (40 L; 12°C ± 1°C) and, once exogenous feeding commenced, were given a continuous supply of appropriately sized commercial trout food (Skretting, the Netherlands) via an automatic feeder.After 8 weeks, fish were hand fed twice daily, and the water volume in the tanks was increased (400 L; 12°C ± 1°C).Tanks were cleaned and siphoned daily to remove waste and any uneaten food.Before the challenge experiment, a subset of fish from each species was cultured to screen for bacterial infections (16), including those caused by F. psychrophilum, and confirmed to be bacterial infection free.

Flavobacterium psychrophilum inoculum preparation for shedding experiment
Flavobacterium psychrophilum variants US19, US62, and US87 were revived from cryostock and verified to be pure cultures as detailed in Preparation of mock water samples containing Flavobacterium psychrophilum section.Each F. psychrophilum variant was inoculated into 250 mL of FPM-A broth and incubated with constant shaking (180 rpm) for 48 hours at 15°C.Bacteria were harvested via centrifugation (2,571 × g, 15 minutes) and adjusted to an optical density at 600 nm (OD 600 ) corresponding to 2.0 × 10 8 cfu/mL using sterile 0.65% saline.Concentrations of each F. psychrophilum variant were determined as detailed in Preparation of mock water samples containing Flavobacterium psychrophilum section.

Intramuscular challenge of fish
Each F. psychrophilum variant was inoculated into the salmonid species it is associated with, according to MLST.Thus, US19 was inoculated into coho salmon, whereas US62 and US87 were inoculated into Atlantic salmon and rainbow trout, respectively.
Atlantic salmon (n = 10; 8 months old; mean weight 18.1 g), coho salmon (n = 10; 8 months old; mean weight 20.5 g), and rainbow trout (n = 10; 8 months old; mean weight 25.1 g) were anesthetized in sodium bicarbonate-buffered (200 mg/L) tricaine methanesulfonate (MS-222; Syndel) at a concentration of 100 mg/L.Then, fish were intramuscularly injected at the base of the dorsal fin with a 50-µL volume of F. psychro philum, equating to a dose of 10 5 cfu/g, and then placed into aerated flow-through glass experimental aquaria (37.85 L; n = 2 aquaria per species, n = 5 fish per aquarium) supplied with ultraviolet light-treated, sand-filtered well water (12°C ± 1°C).Control fish (n = 5 per species, in duplicate aquaria) were treated identically except they were intramuscularly injected with an equal volume of 0.65% saline.The challenge experiment was conducted in accordance with the MSU-Institutional Animal Care and Use Commit tee (AUF:201900312).

Sampling of water containing live and dead fish
All live fish in a replicate experimental aquarium (i.e., main aquarium) were net transferred to a clean non-flow-through glass aquarium (i.e., shedding aquarium; 9.46 L) containing 3,000 mL of fresh, ultraviolet light-treated, sand-filtered well water (12°C ± 1°C) with constant aeration.The shedding aquarium was placed inside a larger, opaque, plastic aquarium that had flow-through water to maintain a water temperature of 12°C ± 1°C in the shedding aquarium; the plastic aquarium also had an opaque cover to minimize light-induced stress.After 1 hour, all fish were removed from the shedding aquarium and transferred back to the main aquarium.A 50-mL water sample was collected using a sterile 50-mL conical tube and then processed immediately as detailed in Bacterial DNA extraction from water section.Water sampling of live fish (including negative control fish) occurred on every other day for the first week, twice per week during the second and third weeks, and then once a week until the end of the experiment (i.e., 4 weeks without detection of F. psychrophilum).
Up to two dead fish per replicate aquarium (i.e., ≤4 dead fish per species) were net transferred into a new flow-through glass aquarium (37.85 L, n = 1 dead fish per aquarium) supplied with ultraviolet light-treated, sand-filtered well water (12°C ± 1°C).After 1, 3, 5, 7, 14, 63, and 98 days post death (PD), fish were transferred to individual shedding aquaria and treated/sampled identically to the live fish.Fish that died that were not used for determining F. psychrophilum shedding rates were necrop sied and clinically examined, and multiple tissues (e.g., external ulcers and kidney) were bacteriologically analyzed for F. psychrophilum on FPM-A.To disinfect shedding aquaria between samplings, aquaria were completely immersed in a 10% (vol/vol) bleach solution for ≥10 minutes, rinsed thoroughly with pathogen-free water, and then air dried.

Determination of Flavobacterium psychrophilum shedding rate from live and dead fish via qPCR
Flavobacterium psychrophilum shedding rates were determined via qPCR (Reaction mixture and cycling parameters section).Briefly, each 96-well qPCR plate consisted of triplicate reactions of eight qPCR standards (10 8 gene copies to 10 1 gene copies), duplicate reactions of template DNA, triplicate reactions of no-template control (e.g., sterile, nuclease-free water), and triplicate reactions of IPC amplification control (e.g., 1,000 copies of Xeno IPC).The shedding rate of F. psychrophilum from infected fish is reported as F. psychrophilum cells shed per fish per hour in 1 mL of water (i.e., F. psychrophilum cells/fish/hour) ( 14) and was calculated using the following formula: F .psycℎropℎilum cells/fish/hour = Mean qPCR gene copies × DECF × 3000 mL Number of fish in aquarium

qPCR standards
All qPCR standards, which spanned nine orders of magnitude (10 8 gene copies/reac tion to 1 gene copy/reaction), successfully amplified.Linear regression of C q values vs the log 10 target gene demonstrated acceptable correlation (R 2 = 0.999 ± .001) with a slope and efficiency of −3.23 ± 0.01 and 104.01%± 0.53%, respectively.The assay was repeatable within and between runs, as CV values were <2.5% within the linear range (10 8 gene copies/reaction to 1 gene copy/reaction) of the standard curve (Table 1).
Consequently and in accordance with Standish et al. (31), these standards were used in all subsequent qPCR assays.

Flavobacterium psychrophilum DNA extraction efficiency and qPCR limit of detection and quantification
Mean F. psychrophilum concentrations of the nine mock water samples ranged from 1.00 × 10 8 to 1.00 × 10° cells/mL and were compared to the mean qPCR-determined concentrations (Table 2).Mean DNA extraction efficiency ranged from 3.63% to 99.70%, with a median of 14.03% (Table 2); therefore, the DECF applied to all qPCR-derived concentrations was 7.128 (100/14.03)(35).
The assay LOD and LOQ were calculated as 30.94 ± 6.84 cells/mL, whereby the DECF (7.128) was multiplied by the lowest mean qPCR-derived F. psychrophilum concentration meeting the qualifications for the LOD and LOQ (e.g., both 4.34 ± 0.96 cells/mL; Table 2).Additionally, amplification was achieved in 40% of the replicates at the next lowest concentration, which corresponded to 13.68 ± 7.09 cells/mL after correction for DNA extraction efficiency (1.92 ± 0.99 cells/mL × 7.128).
The IPC DNA, which was added at the beginning of the DNA extraction process, successfully amplified in all replicate reactions of all but the most concentrated mock water sample (Table 2), which according to the VetMAX Xeno user guide was most likely caused by high target concentration.Nonetheless, of the eight mock water samples that had successful IPC amplification, C q values ranged from 30.12 to 35.29, and all replicates were highly consistent (e.g., CV values ranged from 0.36% to 1.17%; Table 2).

Shedding rates of Flavobacterium psychrophilum from live fish
Following intramuscular injection of 10 5 cfu/g of F. psychrophilum, all tested species (e.g., Atlantic salmon, coho salmon, and rainbow trout) shed F. psychrophilum into the water over multiple days and/or prior to the occurrence of any mortality (Fig. 1A through C).
Live coho salmon began shedding F. psychrophilum into the water at a rate of 1.04 × 10 5 cells/fish/hour 1 day after inoculation.Mortality began quickly in this species (e.g., on day 2) and rapidly reached 100% by day 7.The highest detected F. psychrophilum shedding rate of live coho salmon (e.g., 7.39 × 10 7 cells/fish/hour) occurred on day 5 (Fig. 1C).a Mean, standard deviation, and coefficient of variation (CV) of the cycle threshold (C q ) values are shown.To determine intra-assay variation, the target gene was serially diluted in 10-fold increments over nine orders of magnitude (i.e., 10 8 gene copies/reaction to 1 gene copy/reaction), and eight replicate reactions of each dilution were PCR amplified and quantified.Inter-assay variation was determined by repeating the qPCR run on a separate plate using an identical number of replicate reactions.The assay was repeatable within and between runs, as CV percentages were <2.5 across the linear range (31).Control fish did not shed F. psychrophilum nor did they experience any mortality (data not shown).Likewise, inhibition of gene target amplification was not observed among water samples originating from the negative control or F. psychrophilum-exposed fish, as evidenced by all IPC C q values falling within 28-34.

Shedding rates of Flavobacterium psychrophilum from dead fish
Atlantic salmon, coho salmon, and rainbow trout shed F. psychrophilum into the water from 1 day PD to the end of the 98-day experiment (Fig. 2A through C).Throughout the experiment, fish underwent post-mortem decomposition (Fig. 3A through C).Because of extensive decomposition after day 98, sampling ceased.

Infection status in salmonids challenged with Flavobacterium psychrophilum
Flavobacterium psychrophilum was recovered in a pure form and as perfuse lawns (i.e., colony-forming units were too numerous to count) from external lesions (e.g., muscle ulcerations) and the kidneys of all dead Atlantic salmon, coho salmon, and rainbow trout.Flavobacterium psychrophilum was not recovered from the kidneys of any surviving Atlantic salmon (n = 8 fish; 49 days post inoculation) or rainbow trout (n = 1 fish; 63 days post inoculation).However, F. psychrophilum was recovered in a pure form and as a perfuse lawn from the eye of the only surviving rainbow trout.Because all coho salmon died after 7 days, no survivors could be cultured.

DISCUSSION
For the first time, data on F. psychrophilum shedding dynamics (e.g., time to shedding, shedding rate, and duration in live and dead fish) in Atlantic salmon and coho salmon, which are two of the most BCWD-susceptible salmonid species (3,6,7), have been elucidated.Addressing this knowledge gap was essential, as several studies showed that BCWD epizootics in Atlantic salmon and coho salmon are instigated by phenotyp ically distinct F. psychrophilum variants (14,19,42).Moreover, these F. psychrophilum variants differ from those affecting rainbow trout (15,21,24), the sole species our entire understanding of F. psychrophilum shedding dynamics is based upon.Although some commonalities in shedding dynamics were observed among Atlantic salmon, coho salmon, and rainbow trout, multiple differences were also apparent.Dead Atlantic salmon, rainbow trout, and coho salmon were found to shed F. psychrophilum cells at rates up to 5.4-, 8.5-, and 171.8-fold greater than their living counterparts and did so for extended periods (e.g., up to 77, 63, and 93 days longer).This is the first time that these comparisons have been made among Atlantic salmon and coho salmon, demonstrating that these species, in addition to rainbow trout, are efficient F. psychrophilum shedders.Madetoja et al. ( 14) also found dead rainbow trout shed at higher (e.g., up to ~100-fold) rates and for a longer duration (e.g., 59 days longer) than living rainbow trout.The difference in shedding rate among live and dead rainbow trout observed by Madetoja et al. ( 14) is substantially greater than the 8.5-fold difference found herein for rainbow trout, while differences in shedding duration were similar (59 vs 63 days) (14).Taken together with the findings in Atlantic salmon and coho salmon, results suggest that some F. psychrophilum variants may be more efficiently shed by dead fish than others.Nevertheless, dead F. psychrophilum-infected fish clearly pose a significant risk for disease perpetuation within fish farms and hatcheries, underscoring the importance of implementing management strategies that aim to remove dead fish from rearing units quickly.Like F. psychrophilum, higher shedding rates in dead vs live salmonids have been noted in other fish pathogenic Flavobacterium species (e.g., F. columnare, a cause of columnaris disease) (43,44) as well as Aeromonas salmonicida subspecies salmonicida (e.g., the cause of furunculosis) (45), highlighting dead fish as potential transmission risks for multiple bacterial fish pathogens.
Despite some similarities, multiple differences in F. psychrophilum shedding dynamics by host species and variant were also observed.For example, on day 11, live rainbow trout were shedding 2,923-fold more F. psychrophilum cells per hour compared to live Atlantic salmon.This difference continued to increase through day 21, which was the last day of F. psychrophilum detection among Atlantic salmon.Meanwhile (i.e., on day 21), F. psychrophilum shedding rates remained high among rainbow trout (e.g., 5.98 × 10 7 cells/ fish/hour), and this species continued to shed for another 2 weeks.Adding a further layer of complexity, F. psychrophilum shedding rates among live Atlantic salmon and rainbow trout tended to peak on and/or near days with mortality and could possibly suggest that risk of transmission may vary by F. psychrophilum variant.For example, Atlantic salmon infected with US62 (ST277, in CC-ST232) died over 3 days, whereas rainbow trout infected with US87 (ST275) mostly died over 11 days (i.e., a greater than threefold longer period).Madetoja et al. (14) infected rainbow trout with a different F. psychrophilum variant and observed that fish died over a 4-day period.Therefore, it appears that some F. psychrophilum variants pose a greater transmission risk among live fish compared to others.
The observed differences in shedding dynamics across F. psychrophilum variants are likely influenced by pathogen and/or host factors.In this study, US87, identified in reference (46) as molecular serotype 2 (nearly equivalent to conventional serotype Th) (47,48), showed a protracted shedding period in rainbow trout and persisted systemi cally in a survivor (e.g., for at least 63 days), suggesting that the rainbow trout immune system may struggle in eliminating this variant.In the context of a fish farm or hatchery, this outcome could affect F. psychrophilum transmission dynamics and increase the number of subsequent infections (i.e., the basic reproduction number, R 0 ) (49).US62 also exhibited prolonged shedding herein, albeit to a lesser extent than US87, but appeared to be successfully cleared by Atlantic salmon at the end of the experiment.This F. psychrophilum variant belonged to molecular serotype 1 (nearly equivalent to conventional serotype Fd) (46)(47)(48), which is less common in Atlantic salmon (4) and thus possibly less capable of circumventing this species' immune response, possibly helping to explain the shorter duration of shedding and lack of recovery from survivors.US19 was highly virulent herein, despite using an identical dose, and caused mortality before a rigorous adaptive immune response could be mounted and additional shedding data collected.Whether the shedding dynamics of these variants are representative of other F. psychrophilum variants affecting Atlantic salmon, coho salmon, and rainbow trout should be further investigated.
In addition to elucidating several aspects of F. psychrophilum shedding dynamics in three salmonid species, this study also built upon the previous work of Marancik and Wiens (30) to optimize their qPCR assay for detection and quantification of F. psychrophilum in water.Originally, Marancik and Wiens (30) described this assay for the detection/quantification of F. psychrophilum in spleen tissue, with an LOD of 3.1 genome units per reaction and LOQ of ~486 colony-forming units.Herein, this assay was further optimized to quantify F. psychrophilum from filtered water, with an LOD/LOQ of 30.94 ± 6.84 cells/mL.The sensitivity of the assay could potentially be improved by filtering a larger water volume and/or by increasing sample volume per reaction.Indeed, even a 50-mL water volume containing 10 8 F. psychrophilum cells/mL filtered quickly (i.e., no indication of filter fouling).Strepparava et al. ( 29) also designed a F. psychrophilum-spe cific qPCR to quantify F. psychrophilum from water samples, and although the LOD was similar (e.g., 66 cells/mL), the LOQ was >100-fold higher (e.g., 3,300 cells/mL).Moreover and in the hands of the study authors and at least two other laboratories, specificity issues were observed with this qPCR assay (unpublished data), and so, alternatives were sought.An additional improvement for the qPCR assay herein was the addition of an internal positive control, which allowed for monitoring of PCR-inhibition, a known source of "false negatives" with molecular assays (50).Moving forward, this qPCR assay will be instrumental to future studies assessing F. psychrophilum shedding dynamics and could have application to F. psychrophilum detection in environmental field settings.
Prior to this study, knowledge of F. psychrophilum shedding dynamics was limited only to rainbow trout and a single F. psychrophilum variant.Herein, dead Atlantic salmon, coho salmon, and rainbow trout were shown to shed F. psychrophilum at higher rates than their living counterparts and for at least several months, thereby potentially posing great transmission risk.Therefore and with the insight provided by a recent F. psychrophi lum transmission model (51), it remains critical for personnel raising fish to remove dead, F. psychrophilum-infected fish from rearing units quickly and frequently.Given that F. psychrophilum is now well recognized as a genetically diverse pathogen (15,17,23,24) of varying phenotypes that can affect salmonid hosts differentially (46) in conjunction with the differential shedding results by variant/host species herein, future studies should evaluate the risk of transmission to multiple salmonid species.

FIG 3
FIG 3 Representative images of dead fish used in Flavobacterium psychrophilum shedding experiment.(A) Coho salmon (Oncorhynchus kisutch) 12 days post death.(B) Rainbow trout (O.mykiss) 25 days post death.(C) Coho salmon 98 days post death.Note the yellowish discoloration present on fish.

TABLE 1
Repeatability of Flavobacterium psychrophilum Marancik and Wiens qPCR assay a

TABLE 2
Evaluation of Flavobacterium psychrophilum DNA extraction procedure from water using qPCR c,d

TABLE 3 Mean
Flavobacterium psychrophilum shedding rates (cells/fish/hour) ± standard deviation of dead Atlantic salmon, coho salmon, and rainbow trout on each sampling day a a Number of fish sampled (N) is to the left of the host species name.